Mechanisms for the paradoxical resistance to d-tubocurarine during immobilization-induced muscle atrophy.

This study investigated whether immobilization-induced hyposensitivity to d-tubocurarine (dTC), up-regulation of acetylcholine receptors (AChRs) and changes in fiber size and motor endplate size persist indefinitely and whether they are causally related. Unilateral disuse of the tibialis muscle was produced in adult rats by pinning the knee and ankle joints at 90 degrees flexion. The contralateral unpinned and a separate group of sham-pinned legs served as controls. After 7, 14 or 28 days of disuse, the in vivo dose of dTC that produced 50% depression of nerve-evoked twitch (ED50) in the tibialis muscle increased 3.0-, 3. 2- and 2.1-fold (P < .05), and membrane AChRs increased 6.0- (P < . 05), 6.3- (P > .05) and 1.2-fold (P > .395) relative to control, respectively. Disuse caused muscle fiber atrophy (P < .01) but did not affect endplate size. Hence, the ratio of endplate size to fiber size increased. There was a transient increase in gene expression of all (including de novo expression of the gamma) subunits of the AChR, peaking at day 7 and returning to normal by day 28 of immobilization. The ED50 of dTC correlated directly with AChRs (R2 = 0.51; P < .0001) or the ratio of endplate size to fiber size (R2 = 0. 30; P < .001), and inversely with fiber size (R2 = 0.43, P < .0001). It is proposed that acting together, but not singly, the changes in AChRs, fiber size and relative endplate size contribute to the magnitude and time course of the resistance to dTC produced by chronic disuse.

Na+ channel and acetylcholine receptor changes in muscle at sites distant from burns do not simulate denervation.

Muscle weakness and aberrant responses to neuromuscular relaxants after burn injury are associated with upregulation of acetylcholine receptors (AChRs). Typically, these functional, pharmacological, and biochemical changes occur after denervation, in which transcriptionally mediated qualitative changes in AChRs and Na+ channels and of myogenic regulatory proteins MyoD and myogenin also occur. This study in rats, by an examination of changes in the above-enumerated proteins or their transcripts in the gastrocnemius muscle distant from the burn, verifies whether a denervation-like state exists after burns. Scatchard analysis of [3H]saxitoxin binding revealed no changes in the affinity (K(d)) and total number (B(max)) of Na+ channels between control and burn-injured animals at both 7 and 14 days after injury. The mRNA levels of the immature proteins, SkM2 of the Na+ channels and the gamma-subunits of AChRs, the increase of which is pathognomic of denervation, were assessed by Northern analysis and were unchanged. The transcripts of mature Na+ channels, SkM1, were significantly increased at day 14 after the burn (1.24 +/- 0.10 in burn-injured vs. 1.06 +/- 0.12 in sham animals, arbitrary units, P = 0.006). Although MyoD levels were increased in burn-injured animals at 14 days (0.21 +/- 0.02 vs. 0.15 +/- 0.07 arbitrary units, P = 0.05), myogenin levels were unaltered. The absence of changes in AChR transcripts, including alpha-, delta-, and gamma-subunits, indicates that the upregulation of AChR in burns is not transcriptionally mediated. The unaltered levels of transcripts of myogenin, SkM2 of Na+ channels and gamma-subunit of AChR, confirm that there is no denervation-like prejunctional (nerve-related) component to explain the muscle weakness or the upregulation of AChRs at sites distant from burns.

Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms.

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have > or = 93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 micrograms/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.

Calcium stimulates ATP-Mg/Pi carrier activity in rat liver mitochondria.

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.

Neonatal hypoxia or maternal diabetes delays postnatal development of liver mitochondria.

The matrix adenine nucleotide pool size of rat liver mitochondria was low at birth (2.6-3.0 nmol ATP + ADP + AMP/mg mitochondrial protein). After parturition, the pool size was increased by 50-75% within 1 h, which was sufficient for full development of state 3 respiration rates. The adenine nucleotide pool size continued to increase to 100-150% of the value at birth by 2-3 h postnatal. The ATP/ADP ratio in isolated mitochondria also increased postnatally, to about double the value at birth by 3 h. There were no matrix volume changes over this postnatal period, so the increased ATP + ADP + AMP pool size and the increased ATP/ADP ratio together inferred an overall increase of about 5-fold in the matrix ATP concentration under aerobic conditions. The postnatal uptake of adenine nucleotides into mitochondria occurred at a slower rate in newborns that were hypoxic (11% 02) and in newborns of diabetic mothers (diabetes induced on day 5 of gestation by streptozotocin injection). The normal increase in matrix ATP content is responsible for the rapid stimulation of pyruvate carboxylation (an ATP-requiring matrix reaction) and this in turn contributes to the rapid postnatal onset of gluconeogenesis. The results suggest that delayed adenine nucleotide uptake into liver mitochondria may retard initiation of gluconeogenesis in newborns experiencing hypoxia, as in respiratory distress or in newborns of diabetic mothers. We speculate that this mechanism contributes to the persistent hypoglycemia that is typical of these at-risk newborns.

Mitochondrial function after acute alteration of the endogenous insulin-to-glucagon ratio.

Mannoheptulose (2g/kg i.p.) increases serum glucagon and decreases serum insulin via its effect on pancreatic islet cells. These changes in endogenous hormone status had effects on rat liver mitochondria that were comparable to the effects of injecting porcine glucagon (0.5 mg/kg i.p.). Mitochondrial adenine nucleotide content was increased 38 or 39% by mannoheptulose or glucagon respectively, citrulline synthesis by 165 or 193%, pyruvate carboxylation by 113 or 135%, coupled respiration by 34 or 42%, and uncoupled respiration by 40 or 54%. We conclude that the reciprocal changes in endogenous insulin and glucagon brought about by mannoheptulose offer a useful and interesting alternative to glucagon injection for studying the effects of these pancreatic hormones on liver mitochondria.